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Title Larval Metamorphosis of the Sea Urchins, Pseudocentrotus depressus andAnthocidaris crassispina Induced by Microbial Films

Author(s) Siti Akmar Khadijah binte Ab Rahim

Citation (2004-03-31)

Issue Date 2004-03-31

URL http://hdl.handle.net/10069/20898

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　　　　　LarvalMetamorphosisoftheSeaUrchins，

鳶鰯・・8伽伽s吻・θ5s麗5and14励・・∫伽s徽5s棚nα

　　　　　　　　　　InducedbyMiαobialFilms

December，、2003

G㎜U紐ESCHOOLOFSCIENCE州pTECHNOLOGY

　　　　　　　　　　NAGASAKI　UNIVERSITY

　　　　　　　　　　　　　　　　　JAPAN

SITIAKMARKHADIJAH箪INTIABRAHIM

Larval Metamorphosis of the Sea Urchins,

Pseudocentrotus depressus and Anthocidaris crassispina

Induced by Microbial Films

A dissertation submitted in partial fulfillment of the requirement

for the degree of

Doctor of Philosophy in Marine Science

by

SITI A~MAR KHADIJ~AH~~ BINTl AB ~HIM

Approved by the Research Advisory Committee

Prof. Hitoshi Kitamura

Course Supervisor & Chairperson

Prof. Yuji Fujita prof. Kenji Hara

December, 2003

Graduate School of Science and Technology

Nagasaki University, Japan

ACKNOWLED GEMENTS

I would like to express my sincere thanks and gratitude for help received

during this work to . . .

' My honorable research supervisor Prof. Hitoshi Kitamura, for his

able guidance and constructive criticisms during the course of this

mvestrgation.

e Ministry of Education, Science, Sports and Culture of Japan

(MONBUSHO), for awarding the scholarship.

' Prof. ~Y~uji Fujita and Prof. Kenji Hara, for their valuable advice and

suggestions as referees for doctoral dissertation.

' Dr. Cyril Glenn Satuito, for his kind ~elp and scholastic advice in

writing up this thesis.

' Prof. Toru Takita, for his kindness to introduce me with my research

supervrsor.

e Universiti Malaysia Sarawak (UNIMAS), fbr granting me the study

leave to conduct the present research.

' My lab-mates: Mrs. Lee, Mr. Fujikawa, Mr. Jouuchi, Mr. Shiraishi,

Mr. Taniguchi, Miss Kouda, Miss Yokoyama and other students of

Laboratory of Biological Environment Science for their kind help

during my work.

e Kiwakai Ai_ 'kido Club members.

' Kokusai Kouryuu Juku (Chikyukan) members.

●●

細岨andfriend亀f・rtheirsupP・rtandunderstanding

Final1第Ials・wisht・c・nveymydeepsense・fgratitudet・my

bel・vedfamily；with・uttheirenc・uragementandblessin＆this

achievementwould　not　have　been　possible．

to my parents. . .

CONTENTS

Page

English Abstract ------ __ i

Japanese Abstract ------ __ v

List of Figures ------ __ vii

Chapter - I. ueneral mtroductron ---- 1

Chapter - II. Larval metamorphosis of the sea urchins,

Pseudocentrotus depressus and Anthocidaris

crasslspma m response to microbial films formed

in the sea and diatom-based films formed in the

mass-production tanks -------------------------------_ 6

Chapter - 111. The biological factors of diatom-based film for

larval metamorphosis of the sea urchins,

Pseudocentrotus depressus and Anthocidaris

crassrspma ---- ____ 26

Chapter－IV＝

Ch叩ter」V

Refbrences

Characteristic　ofthe　chemical　cue　in　diatom．based

films　for　larval　metamorphosis　ofthe　seaurchins，

鳶εμ40cεn∫7伽s吻rεs5μ5andオn孟hoc∫伽∫s

C7αSS嗣ρ必α　一一一一一一一一一一一一一一一一一一一一一一一一一一一一＿

General　discussion　and　conclusions　一．舳一一一一．一．一．一一

45

59

66

AB STRACT

Most of marine benthic invertebrates possess planktonic larvae,

which gain the ability to select a suitable habitat for settlement and

metamorphose into juveniles during their competent stage. The

selection of substrate for larval metamorphosis is influenced by chemical

cues from microbial ~ilms, algae (heterospecific) and conspecific adults;

and also by physical factors. In the western part of Japan, diatom-based

film grown on plastic plates is used to induce larval metamorphosis of

sea urchin during the mass-production of juveniles. However, the role

of periphytic diatoms and bacteria co-existing in the films is poorly

understood. Therefore, this study focused on the larval response of two

commercially important species of sea urchin, Pseudocentrotus

depressus and Anthocidaris crassispina to microbial films developed in

the sea, diatom culturing tanks and laboratory conditions.

In Chapter - I, the relationship between microbial film and larval

metamorphosis of marine invertebrates especially of fisheries important

species were outlined. The role of microbial film in aquaculture for

mass-production of sea urchin juveniles was focused in this study.

In Chapter - II, the induction of larval metamorphosis of two

species of sea urchin by micyobial films formed in the sea and in the tank

(5-tonne) for sea urchin juvenile mass-production was studied for several

years. Films formed in the sea showed that the metamorphosis (%) of P

i

depressus increased gradually in accordance with the immersion period

of film formed on glass slides, whereas the larval metamorphosis of A.

irassispina showed a bell shaped response curve. In the tank, although

the diatom-based films showed a low inducing activity for larval

metamorphosis of A. crassispina, the metamorphosis of P depressus

larvae increased linearly in accordance with the diatom density. These

suggest that diatom-based films could promote the larval metamorphosis

of P depressus, but are less important in A. crassispina . Based on other

reports, I concluded that the high response of the former towards

nucroDral nlm tnan tne tatter rs due to the di~tt~erence in their natural

habitats where juveniles of P depressus are found in deep rocky subtidal

areas while ofA. crassispina live in shallow intertidal coralline algae bed.

Microbial films on rock surfaces act as nietamorphosis inducer for larvae

of P depressus, while the cue from algae induces larval metamorphosis

ofA. crassispina .

Chapter - 111 investigated the roles of diatoms and bacteria which

comprised laboratory-cultured diatom-based films (several diatom

species including bacteria) in the induction of metamorphosis in sea

urchin larvae. These diatom-based films induced larval metamorphosis

in both sea urchins, but the response rate was higher in P depressus.

Diatoms collected on the glass-fiber filters induced metamorphosis in

larvae of both species, although the inducing activity was significantly

less than that of the diatom-based films. No larva metamorphosed on

ii

bacterial films that formed from the filtrates, suggesting that bacteria

alone cannot induce metamorphosis and that diatoms play a major role in

the induction. Antibiotic treatment during assay reduced the inducing

activity in diatom-based films, even though the treatment did not control

bacterial growth. None of five isolated species of periphytic diatom

induced larval metamorphosis by itself and this suggests that no certain

diatom species was directly involved as an inducer. For diatom-based

film cultured in the presence qf germanium dioxide (Ge02) or antibiotic,

both species of sea urchins showed a similar response, in which

reduction in diatom and bacteria density resulted in a decrease in the

original inducing activity. There seems to be a synergistic effect

between diatom and bacteria in inducing larval metamorphosis.

Chapter - IV attempted to elucidate the characteristic of chemical

cue in laboratory-cultured diatom-based film. Films subjected to

treatment with 45'C heat, 0.1 N HCI and glutaraldehyde (5%) were no

longer inductive for either sea urchins, while those films treated with

40'C heat or EtOH (5%- and 10%-EtOH) showed a significant reduction

in the inducing activity. However, Iectin (LCA, SBA and WGA)

treatments had no effect. It i~ suggested that metamorphosis-inducing

cue(s) of live diatom-based films was unstable and sugar-related

compounds do not play a main role in the metamorphosis.

Chapter - V concluded that (1) microbial films acted as an inducer

for larval metamorphosis for both species of sea urchin although the

iii

response was different for each species and correlated with their natural

habitat. (2) It seems that diatoms and bacteria in the diatom-based

films produced a synergistic effect for induction of metamorphosis in sea

urchin larvae. (3) The metamorphosis-inducing cue(s) in diatom-based

films was unstable and sugar-related compounds do not play a main role

in the metamorphosis. This study provides initial information on the

role of microbial films on induction of larval metamorphosis in two

species of sea urchins, P depressus and A. crassispina and may improve

the techniques of juveniles mass-production of fisheries important

species of marine invertebrates especially sea urchins and other

echinoderms. Moreover, the identification of the chemical structure of

the cues and the receptor sites in the larva are important for

understanding the mechanism and chemical ecology of metamorphosis

in sea urchin larvae.

iv

La「va1弊etam・rphgsis・ftheSeaUrchins，P躍・cεn繊s吻ε5s麗sand。4n孟hoα伽7∫sc7αs3砂∫nαInducedbyMicrobialFilms

長崎大学大学院生産科学研究科

シティ・アクマル・カディジャ・ビンティ・アブル・ラヒム

（SitiAkmar　KhadijahbinteAb　Rahim）

　海洋無脊椎動物の多くは浮遊幼生を経て、底棲性の成体になる。幼生から成体

への変態には、種々の基質と接触することが重要であり、この際に化学的な刺激

　（ケミカルシグナル）や物理的な刺激を感受し、変態に至るものと考えらる。基

質としては・海藻や微生物フィルムなどが知られており、西日本におけるウニ種

苗生産でも、波板に付着珪藻フィルムを繁殖させて幼生の変態誘導を行ってい

る・しかし・微生物フィルムによるウニ幼生の変態機構についての知見は少ない。

そこで本研究では・西日本で水産上重要なアカウニ（」附θαの。θ四伽o云α5

砲ρ兜33α3）及びムラサキウニ（。4滋hoα幽万5α25磯ρ血8）幼生を対象として、

海域、大型水槽および室内実験にて、微生物フィルムと変態との関係を様々な観

点から追究した。

　第1章では、ウニ幼生の変態と微生物フィルムとの関係について研究の概要を

まとめた。

　第2章では、海域およびウニ種苗生産水槽（5トン）において形成された微生

物フィルムとアカウニ、ムラサキウニ幼生の変態誘起との関係を数年間にわたり

調べた。その結果、海域フィルムでは、アカウニの場合、垂下日数に従い変態率

が上昇したが1ムラサキウニでは山形（ベルシャープ）の傾向となった。種苗生

産水槽では、付着珪藻主体のフィルムが形成されるが、アカウニでは、珪藻密度

と変態活性とには明瞭な正の相関関係が認められた。しかし、ムラサキウニでは

全体に活性が認められない傾向となった。既報との関連から、アカウニは微生物

フィルムに反応し、ムラサキウニ幼生は海藻との関係で変態するものと結論され

た。

　第3章では、付着珪藻フィルム（多種の付着珪藻および付着細菌）を室内にお

いて培養し、変態活性を確認すると共に、付着珪藻と付着細菌の役割について検

討した。ガラス繊維濾紙により珪藻と細菌とを大まかに分離したところ、珪藻側

V

に活性が認められた。先の付着珪藻フィルムから5種類の付着珪藻を単離した

が、これらに変態活性は認められなかった。また、珪藻の増殖阻害剤である二酸

化ゲルマニュームを添加した場合、および抗生物質を添加して珪藻フィルムを形

成させた場合は、両種共に、抗生物質添加でより大きく活性炉低下した。以上の

ことから、珪藻と綿菌とは各々単独では誘起活性が低く、両者が混合して活性が

発現してくるものと推察した。

　第4章では、室内培養の付着珪藻フィルムについて、熱（300C－450C）、EtOH

（1％、5％及び10％）、塩酸（0．1N及び1．O　N）の各処理、さらにはグルタール

アルデヒド固定を行ったところ、両種共に、活性は400C、EtOH（10％）、塩

酸（0．1N）処理で著しく低下した。またレクチン（LCA，SBA，WGA）添加で

は活性に影響がなかった。これらより培養珪藻フィルムの変態誘起物質は、著し

く不安定な物質であると推定された。また珪藻や細菌などの生死が活性発現に大

きく関係していることも示唆された。

　以上のように、アカウニ及びムラサキウニ幼生に対して、両種で感受性は異な

るものの、微生物フィルムが変態にとって重要であることを明らかにした。次に

微生物フィルムの主な構成生物である付着珪藻と細菌とは共存して変態に関わ

るものと考察した。またフィルムの持つ変態誘起物質は化学的に不安定な物質で一

あると推定した。これらの知見は、ウニ類を含めた水産無脊椎動物の種苗生産技

術の改良、さらには海洋生物の変態機構の更なる解明につながるものと思わ液

る。

vi

LIST OF FIGURES

Page

Figure - 2.1. Methods for the larval culture. ------------------------- 9

Figure - 2.2. Larval assay procedure. ---------------------------_____ 11

Figure - 2.3. Preparation of (a) microbial films in the sea and

(b) diatom-based films in the mass-production

tanks. ---------------------------___________________________ 13

Figure - 2 4 Larval metamorphosis of P d ' ' ' , epressus m response

to microbial films of different immersion period

formed in the sea. --- ____ 16 Figure - 2.5. Larval metamorphosis ofA, crassispma m response

to microbial films of different immersion period

formed in the sea. --------------------___________________ 17

Figure - 2 6 Larval metamorphosis of P d ' ' ' . epressus m response

to diatom-based films of di~erent diatom density

formed in the tank. -------------_________________________ 19

Figure - 2.7. Larval metamorphosis ofA. crassispma m response

to diatom-based films of different diatom density

formed in the tank. ------------__________________________ 20

Figure - 2.8. Larval metamorphosis of two species of sea urchin,

P depressus and A, crassispina in response to

diatom-based films of different diatom density

formed in the tank in May. ---------------------------- 22

vii

Figure - 3.1.

Figure - 3.2.

Figure - 3.3.

r=igure - 3.4.

Figure - 3.5.

Page

Diatom-based film cultured in the laboratory. ------ 28

Preparation of diatoms on GF/C filter and

bacterial film developed from filtrate. --------------- 30

Larval metamorphosis of P depressus in response

to diatom-based films, diatoms on filters, bacterial

films and antibiotics-treated diatom-based films

(0.5 mg ml~1 penicillin G potassium and 0.01 mg ml~1

of streptomycin sulphate) during assay. ------------- 34

Larval metamorphosis ofA. crassispina in response

to diatom-based films, diatom on filters, bacterial

films and antibiotic-treated diatom-based films

(0.5 mg ml~1 penicillin G potassium and 0.01 mg ml~1

of streptomycin sulphate). ----------------------------- 35


to diatom-based films cultured with germanium

dioxide. Ge02 (6.0 ug ml~1) and cultured with

antibiotic mixture (3.0 mg ml~1 penicillin G

potassium and 0.06 mg ml~1 of streptomycin

sulphate). ----------------------------------------------___ 37

viil

Page

Figure - 3.6.

Figure - 3.7.

Figure - 3.8.

Figure - 4.1.

Figure - 4.2.

Figure - 4.3.


to diatom-based films cultured with germanium

dioxide, Ge02 (6.0 ug ml~1) and cultured with

antibiotic mixture (3 .O mg ml~1 penicillin G

potassium and 0.06 mg ml~1 of streptomycin

sulphate). ------------------------------___________________


to five species of periphytic diatom isolated from

diatom-based film. ---------------------------___________

Larval metamorphosis ofA crassis ' ' . pma m response

to five species of periphytic diatom isolated from

diatom-based film. --------------------------____________

Summary of heat and chemical treatments. ---------


to diatom-based films treated with heat (300C, 350C,

400C and 450c). ________________________________________


to diatom-based films treated with heat (300C, 350C,

400C and 450C). ----------------________________________

38

40

41

48

50

51

ix

Figure - 4.4.

Page


to diatom-based films treated with EtOH (1%, 5 %

and 10%), HCI (0.1 N and 1.0 N) and glutaraldehyde

(5%). ---------------------------------------.-------------- 53

Figure - 4.5.

Figure - 4.6.

Figure - 4.7.


to diatom-based films treated with EtOH (1%, 5%

and 10%), HCI (0.1 N and 1.0 N) and glutaraldehyde

(5%). ------------------------------------------------------ 54


to diatom-based films treated with lectins (LCA,

SBA and WGA). -----------------------------------_____ 55


to diatom-based films treated with lectins (LCA,

SBA and WGA). -------------------------------_________ 56

x

CHAPTER - I

GENERAL INTRODUCTION

The life cycle of most marine invertebrates includes a planktonic

larval stage prior to a benthic adult phase. A critical phase during the

larval lifespan occurs when dispersed larvae gain the ability to select a

suitable habitat for settlement and metamorphose into juveniles. This

selection determines the long-term survival and consequently; the larvae

ot' a wide range of marine invertebrates do not settle and metamorphose

unless they encounter specific conditions, ensuring for their growth and

survival (Crisp, 1974; Cameron and Schroeter, 1980; Crisp et al. , 1985;

Snelgrove et al. , 1999).

Typically, settling larvae exhibit a specific searching behavior upon

contact with a suitable cue such as microbial film, macroalgae

(heterospecific) and conspecific adults; and also physical factors for

mstance, water flow, Iight and surface texture (Pawlik, 1992). Many

invertebrate larvae also exhibit the ability to delay metamorphosis until a

suitable substrate is available (Barker, 1977; Pechenik et al., 1993;

Mercier et al., 2000).

Microbial film is typically described as a succession of changes,

beginning with the formation of a layer of organic molecule film

consisting of amino acids, glycoproteins and humic materials, and

1

advancing to the colonization by bacteria, diatoms, fungi and protozoa

(Loeb and Niehof, 1975; Mitchell and Kirchman, 1 984; Costerton et al. ,

1995; Bhosle and Wagh, 1997; Tsurumi and Fusetani, 1998). The

formation of microbial films is a prerequisite for the metamorphosis of

marine invertebrate larvae (Zobell and Allen, 1935; Crisp, 1974; Pawlik,

1992; Johnson et al., 1997; Wieczorek and Todd, 1998).

Cues from microbial films induce larval metamorphosis of

coelenterate (Leitz and Wagner, 1 993), bryozoans (Brancato and

Woollacott, 1982; Keough and Raimondi, 1995), annelid (Kirchman et al.

1982; Unabia and Hadfield, 1999; Lau and Qian, 2001), mollusks

(Kawamura and Kikuchi, 1992; Slattery, 1992; Satuito et al. , 1995,

Robert and Nicholson, 1997) and echinoderms (Cameron and

Hinegardner, 1974; Barker, 1977), but has no effect on barnacles (Maki

et al., 1988).

Anrong echinoderms, it has been demonstrated that the larvae of

starfishes C , oscinasterias calamaria (Barker, 1 977) and Asterias rubens

(Barker and Nichols, 1983), settled on many kinds of hard substrates

covered with microbial films. The recruitment of crown-of-thorns

starfish Acanthaster planci was also associated with the periphytic

bacteria of crustose coralline algae (Johnson et al. , 199la, 199lb;

Johnson and Sutton, 1994). Bacteria-induced metamorphosis of the

sand dollar, Arachnoides placenta, was also reported in beakers used for

larval culture (Chen and Run, 1989). In sea urchins, Cameron and

2

Hinegardner (1974) observed that laboratory-reared Lytechinus pictus

and Arbacia punctulata larvae underwent metamorphosis in response to

microbial film. Similarly; field experiments on larval settlement in

Strongylocentrotus franciscanus and S. purpuratus (Cameron and

Schroeter, 1980; Rowley, 1989) and S. droebachiensis (Pearce and

Scheibling, 1991) demonstrated that surfaces with their microbial film

removed did not induce metamorphosis, whereas settlement did occur on

intact films.

Diatom-based film had been used in aquaculture for producing

juveniles of sea urchin, sea cucumber and abalone to sustain the wild

stock. In Japan, economically important sea urchin juveniles

(Anthocidaris ' ' Hemicentrotus crassl spma, pulcherrimus,

Pseudocentrotus depressus, S. intermedius, S. nudus) are being

mass-produced annually (Kltamura et al. , 1993). A flat or corrugated

plastic plate ("nami-ita") covered with periphytic diatoms in 5- to

15-tonne tanks is the most widespread method used in fish farms for

inducing the settlement and metamorphosis of sea urchi (T n ani and Ito,

1979; Ito, 1984; Ito et al. , 1991; Kawahara, 1996; Kitamura et al., 2000),

sea cucumber (Ito and Kitamura, 1997) and abalone (Kawamura and

Kikuchi, 1992; Kawamura, 1 996) Iarvae.

Despite the fact that success in mass-production of sea urchin

juveniles can be attributed to the successfiil use of diatom-based films as

inducers of metamorphosis in sea urchin larvae, the role of periphytic

8

diatoms and bacteria co-existing in the films is poorly understood. We

need more information on the roles of diatoms and bacteria as inducing

factors, in order to understand the importance of microbial film.

Therefore, this study focused on the larval response of two species of sea

urchin, P depressus and A. crassispina, which are important in Japanese

aquaculture, to microbial films formed in the sea and diatom-based films

formed in the tank used for culturing the periphytic diatoms (Chapter -

II) .

It is also important to understand the roles of diatom and bacteria

in diatom-based t~ilm and~ the mechanism involve in the induction of

larval metamorphosis of these two species of sea urchins. In Chapter -

III, studies were done to elucidate the biological factors in

laboratory-cultured diatom-based films that induce metamorphosis in sea

urchin larvae. We investigated the effect of diatoms and bacteria on

larval metamorphosis using diatom-based films cultured in the presence

of germanium dioxide or antibiotic mixture. In addition, we evaluated

the metamorphosis-inducing activity of five periphytic diatom species

isolated from the films.

In Chapter - IV, attempts were made to elucidate the characteristics

of the chemical cue in laboratory-cultured diatom-based film that

induced larval metamorphosis of P depressus and A. crassispina larvae.

The metamorphosis-inducing activity of film-associated cue was

evaluated after heat, ethanol, hydrochloric acid, glutaraldehyde or lectin

4

treatments．

5

CHAPTER - II

Larval metamorphosis of the sea urchins, Pseudocentrotus depressus

and Anthocidaris crassispina in response to microbial films formed in

the sea and diatom-based fihn formed in the mass-production tanks

II - I Ihtroduction

Larval metamorphosis is a nonrandom process that involves the

surface exploration and the final choice of suitable site is guided by

specific signals associated with a marine substratum (Pawlik, 1992;

Wieczorek and Todd, 1998). It has been observed in fieiu experiments

that echinoderm larvae metamorphosed on microbial films of substratum

and crustose coralline algae (Barker and Nichols, 1983; Johnson et al. ,

199la). Surfaces with their microbial film removed did not induce

larval metamorphosis of the sea urchins Stron8ylocentrotus franciscanus,

S. purpuratus (Cameron and Schroeter, 1980; Rowley, 1989) and S.

droebachiensis (Pearce and Scheibling, 1991).

In Japan, two popular methods are used for the annual

mass-production of five economically important sea urchins juveniles

(Anthocidaris ' ' Hemicentrotus pulcherrimus , crassl spma ,

Pseudocentrotus depressus, S. intermedius, S. nudus) (Kitamura et al. ,

6

1993). That is, competent larvae are induced to metamorphose on flat

or corrugated plastic plates ("nami-ita") covered with periphytic

diatom-based films in the western part of Japan and small discoid green

alga Ulvella lens in the northern region (Takahashi et al., 2002).

The studies mentioned above indicated that microbial films,

especially diatom-based films, have an importaht role in larval

metamorphosis of sea urchins. However, the role of periphytic diatom

and bacteria co-existing in the film still remains unknown. We need

more information on the role of diatom and bacteria as inducing factors

m order to understand the importance of microbial film.

Therefore, this study focused on the larval response of two species

of sea urchin, P depressus and A. crassispina, to microbial films in the

sea and diatom-based films formed in the tanks used for culturing the

periphytic diatoms.

II - 2 Materials and methods

For larval culture of two species of sea urchin, broodstocks of

Pseudocentrotus d epressus were purchased and stocked in the Nagasaki

City Fishery Center, Nagasaki, Japan while that of Anthocidaris

crassrspma were sampled from Nomo Bay, Nagasaki, Japan. Larvae of

P depressus and A. crassispina were cultured in the laboratory to the late

7

eight-armed competent stage with a fully developed echinus rudiment

(Kitamura et al. , 1993) (Fig. 2.1). Since the habitat and spawning

season of these two species differed, comparison on the larval response

was evaluated during May when both larvae were available.

Spawning of the sea urchins was initiated by 0.5 M KCI solution.

Eggs were rinsed with filtered seawater (FSW, WhatmanR glass fiber

filter, GF/C; I .2 um), fertilized with a dilute suspension of sperm and

rinsed several times with FSW. After ca. 20 h, swimming, prism larvae

were transferred to two 30-liter tanks filled with 25 Iiters of FSW

(stocking density ca. I Iarva ml~1). ~1'he culture tanks were continuously

aerated (200 ml min~1) in a dark room. About half of the seawater (10

liter) was renewed every day! Polyethylene nets, with mesh openings

of 111 and 225 um, were used sequentially as the larvae grew to filter off

the larvae. Food for the larvae was added after refilling the tanks.

The diatom Chaetoceros gracilis was used as food for larvae at

concentrations of 0.5xl04 to 3.0xl04 cells ml~1 in the culture tank; the

concentration was increased as the larvae grew. C. gracilis was

propagated under unialgal conditions in 1-liter flat-bottom flasks with

mixture of 900 ml of sterilized FSW, I ml of sodium metasilicate

nonahydrate (45mg ml~1) and 0.5 ml of KW21 medium (Daiichi Seikou

Co., Kumamoto, Japan) at 25'C. The flasks were illuminated at ca.

10,000 Lux and aerated (10 Iiter min~1) continuously. The diatom

increased in number from an initial density of 5.0xl04 cells ml~1 to more

8

Adult sea urchins: Anthocidaris crassispina (Nomozaki)

Pseudocentrotus depressus (Makishima)

Spawning induced with 0.5 M KCl

sperm egg artificial fertilization

prism stage larvae

30-1 tank --~

25 1 FSW

10 1 FSW change/day

<:::~"~:::::: v ~ V ~

~V *" day ~ V ~~V ~"

1~ ooo c ov ~

:~~ ~ ~ vv ~

~~

~v ~ v ~ ation o

I~

Stocking density = I Iarva nil~1

Food = Chaetoceros gracilis

22_250C (A. crassispina)-2 weeks

17-220C (P depressus)-3 weeks

Fig. 2.1. Methods for larval culture.

9

than 1.0xl07 cells ml~1 within I week.

The larvae of P depressus were cultured at 17 to 22'C for ca. 3

weeks from October to March. In addition, competent larvae were also

obtained from the Nagasaki City Fishery Center, Nagasaki, Japan and the

Saga Prefectural Fish Farming Center, Chinze-cho, Saga, Japan in May.

The larval cultures of A. crassispina were done at 22 to 250C for ca. 2

weeks from May to August.

Larval assays for Pseudocentrotus depressus and Anthocidaris

crassispina were conducted 4 to 5 times from October to March and

once m May; and 6 to 7 times from May to August, from 1 997 to 2002,

respectively, In May; we had one chance to conduct the larval assay of

both species of the sea urchins simultaneously. In all assays, 200-ml

glass (PyrexR) beakers filled with 100 ml FSW were used (Fig. 2.2).

Subsequently, thirty 8-armed competent larvae were placed into each

beaker containing a piece of filmed glass slide or filmed beaker. These

beakers were kept in a dark room at 18'C and 22'C for P d . epressus and

A crasslspma respectrvely. Two types of control groups were

prepared in each assay: Iarvae exposed only to FSW, to ensure that the

larvae used were not spontaneously undergoing metamorphosis (negative

control); and larvae exposed to diatom-based films, to ensure that the

larvae used were competent to metamorphose (positive control).

Larvae were observed under a stereoscopic microscope after 24 h.

Larval response was assessed in terms of the proportion of

lO

Assay period P depressus : Oct. - Mar. &~)

A, crassispina : Aug

100 ml FSW

30 Iarvae ' 24 h (dark)

180C: P depressus

220C: A, crassispina

Microbial film (sea) Laboratory grown or diatom-based film (tank) diatom-based film

on glass slide

(38X26 mm)

Metamorphosis (%) = no,ofjuveniles X 100

30

Fig. 2.2. Larval assay procedure.

11

1~

metamorphosed individuals (%), defined as the percentage of individuals

that had undergone full metamorphosis to the juvenile form. All assays

were conducted in three to six replicates.

Microbial film samples in the sea were prepared by immersing

glass slides at Makishima Bay from a raft located near a jetty belonging

to the Nagasaki City Fishery Cepter, Nagasaki, Japan (Fig. 2.3a). Half

portion of glass slides (38x26 mm) were placed in plastic holders and

immersed vertically I .5 m below the sea surface. Immersions were

done almost throughout the year in 1996, 1997 and 1999, with each

immersion periods between I and 25 days. The means of diatom and

bacterial densities at twenty random spots on each glass slide were

recorded using a light microscope at 200x magnification and using the

phase-contrast microscope at 400x magnification, respectively. At the

same time, the genera of diatoms and other components in the film were

recorded.

Simultaneously with this experiment, similar glass slides were

immersed in 5-tonne tanks that contained plastic plates coated with

periphytic diatoms for the mass-production of sea urchin

Pseudocentrotus depressus juveniles (Fig. 2.3b). Each immersion

period was between I and 54 days. Navicula, Amphora, Achnanthes

and Nitzschia were the predominant diatom species, which proliferated

on the plates. Mass-production of sea urchin juveniles was carried out

twice a year (May and October) and the periphytic diatom plates in tanks

12

/ (a) Microbial film (sea)

- Immersion period: 1-25 days

R af t

1.5 m

III

Glass slide

(38X26 mm)

~

Plate

(b) Diatom-based film ("nami-ita" tank)

- Immersion period: 1-54 days

y

7

7

7

y

~

~

~

~

n Nami-ita'f

lll 111

Glass slide

(38X26 mm)

Plate

5-tonne tank

Fig. 23. Preparation of (a) microbial films in the sea and

(b) diatom-based films in the mass-production

tanks.

13

were maintained throughout the year.

To propagate periphytic diatoms on the plates, a flowing seawater

system with aeration was used. The light intensity inside the tanks was

controlled by covering the tanks with black mesh sheets (on a sunny day;

70-90% reduction in light intensity and on a cloudy/rainy day, 50%).

Fertilizers for diatom growth were added to the tank, and pesticides

(0.5-1.0 ppm of trichloroform, which is a safe level for sea urchin

juveniles) were used to eliminate the copepods that graze on the diatom

films. Plates coated with periphytic diatoms were washed with

high-pressure seawater (1-2 times per week) in order to select the highly

adhesive and small type of diatoms. Diatom and bacterial densities on

the glass slides were estimated following the same procedure mentioned

earlier. Microbial films and diatom-based films were then subjected to

larval assays described earlier.

All data are expressed as means (:!:SD) of three to six replicates

from three trials. Data were analyzed using the Pearson correlation test

(P<0.01) for the relationship between larval metamorphosis (%) and

diatom density in the tank film. One-way ANOVA followed by

Tukey's multiple comparison test (a=0.05) was used to compare the

metamorphosis (%) of control and other treatments D 'ff . I erences were

considered significant at P<0.001 .

14

II - 3 Results

Throughout all the experiments, the metamorphosis (%) of

competent larvae of Pseudocentrotus depressus and Anthocidaris

crassispina in clean beakers containing FSW was always O%.

The relationship between immersion period (days) and

metamorphosis (%) of two species of sea urchins on microbial films

formed in the sea is shown in Fig. 2.4 and 2.5. Metamorphosis of P

depressus increased gradually with immersion period (October to May)

(Fig. 2.4). Films immersed between 8 days and 23 days showed more

than 20% metamorphosis. On the other hand, the larval response ofA.

crassispina (Fig. 2.5) followed a bell-shaped curve from May to August.

Metamorphosis was high between 8-day and 15-day films, and the

metamorphosis-inducing activity decreased with immersion period. In

one-third of the total trials, the larvae of A. crassispina did 'not

metamorphose in response to microbial film.

Throughout the experiments in the sea, salinity and temperature at

the immersion site were 30-340/00 and 14-30'C, respectively! During

the rainy season (June to mid-July), the main components of the films

were detritus and silt. In the winter season, periphytic diatoms, such as

Navicula, Nitzschia, Amphora, Achnanthes, Licmophora and Cocconeis,

became predominant and the density reached about l05 cells cm~ .

During other seasons, aside from periphytic diatoms, other organisms

15

Oct.-Jan., May

~ ~~.'

~ ~ ~::

~ o ~ o ~:

1 OO

80

60

JA 4U

20

O

*

A A 11FO

A o. AO 6 A

*

* o A

AA

,

A

JL

e'o

A~ eO

A

A Oct

o Nov

e Dec

c Jan

* May

O 5 1 O 1 5 20 Immersion period (days)

25

Fig. 2.4. Larval metamorphosis of P depressus in response to

microbial films of different immersion period formed

in the sea. Each symbol represents the mean meta-

morphosis percentage of three replicates.

16

1 OO

80 '~, ~~OO

,~, , ea_,u:1 60

o ~l ~*

o ~ An ~! '~ o ~:

20

o

May-Aug.

vV

$e

v~.~

* $$

o $$ * *

v.

v

$$ ve v ~ e * o

'v ov 'vve *e

$e

* May

o Jun

v Jut

O 5 10 15 20 Immersion period (days)

25

Fig. 2.5. Larval metamorphosis ofA. crassispina in response to

microbial films of different immersion period formed

in the sea. Each symbol represents the mean meta-


17

such as protozoa, fungi, algal spores, filamentous blue-green algae and

copepods were also found on the film. In the sea films, we faced a

difficulty in counting the bacterial density accurately by direct counting

method, owing to the thickness of the film.

Half portion of glass slides (38x26 mm) were immersed in the

periphytic diatom plates culturing tanks under controlled conditions with

salinity and temperature of seawater being 30-340/00 and 10-30'C,

' respectively. Diatom density ranged from 103 to 106 cells cm~2

comprising Navicula, Nitzschia, Amphora, Tabularia and Achnanthes.

In determination of the bacterial density, we faced a difficulty owing to

the thickness of the film.

The relationship between the diatom density (cells cm~2) and

metamorphosis (%) of the diatom-based film in the tanks is shown in Fig.

2.6 and 2.7. Metamorphosis of P depressus larvae increased linearly,

with diatom density ranging from 2.0xl04 to 6.0xl05 cells cm~

(Y=10.898lnX-93.205; Pearson coefficient 0.5372, P<0.01) (Fig. 2.6).

The correlation coincided with that for the data of Saga Prefecture (1987;

Fig. 2.6). The experiment in Saga was done inside beakers by inducing

the larvae to metamorphose on corrugated plastic plates, covered with

diatom-based film, which were cut into small pieces. In contrast, the

larval metamorphosis ofA. crassispina was less than 20% in nearly 90%

of the total trials, although the diatom densities were high (Fig. 2.7).

Larvae ofA. crassispina showed lower metamorphosis on diatom-based

18

'~ ~~oe

,J enO

~::

el

O ~O

~:

100

80

60

40

20

O

Oct.-Jan., May

A Y=10.898hlX-93.205 A

r=0.5372 AA AAA

A

A ,A A A A

AAA

~!~

A AA^

A A~~

A

~

A

A

1 03 1 04 1 05 1 06

Diatom density (cells cm~2)

1 07


diatom-based films of different diatom density formed

in the tank. Each symbol represents the mean meta-

morphosis percentage of three replicates. [x] Data

from Saga Prefectural Fish Farming Center (1987).

19

1 OO

80 '

~ ~~"

~ ~ 60 ~:Io

~h

~~ 40

~:

20

O

May-Aug.

Y=2.5188lnX-22.995 r=0.4636

o

oO 'O o O~-

O . er' Oo o O QOO;~~O OCO d 9

9, o

o

,

1 03 1 04 1 05 106


1 07


diatom-based films of different diatom density formed

in the tank. Each symbol represents the mean meta-


20

film compared to P depressus. Moreover, it was also observed that the

larvae of P depressus did not metamorphose on films with a diatom

density of <104 cells cm~ .

During the experiment with the diatom-based film, simultaneous

larval assay of these two species P depressus and A. crassispina, was

carried out in May (Fig. 2.8). As the diatom density increased from

2.0xl04 to 2.0xl05 cells cm~2, the mean metamorphosis rate for the

form~r was 39% and for the latter, 5%. Moreover, a closer correlation

between metamorphosis rate and diatom density was observed in P

depressus (Y=23.707lnX-217.14; Pearson coefficient 0.7066, P<0.01)

than in A. crassispina (Y=3.8753lnX-38.235; Pearson coefficient 0.4802,

P<0.01), which showed a clear difference between the two species in

response to the metamorphosis-inducing activity of diatom-based film.

21

100

80

~ ~~.'

~ "~ 60 ~i

~h

~ 40 ~

20

O

Pseudocentrotus depressus

Y=23.707lnX-217.14 r=0.7066

A A

A

A .A

A o A O A ..･･' ' 6

R;'~ O o

Anthocidaris crassispina

Y=3.8753lnX-38.235 r=0.4802

103 1 04 1 05 1 06


1 07

Fig. 2.8. Larval metamorphosis of two species of sea urchins,

P depressus and A. crassispina in response to diatom-

based films of different diatom density formed in the

tank in May. Each symbol represents the mean meta-


A : P depressus; O : A. crassispina

22

II - 4 Discussion

The induction of larval metamorphosis of most marine

invertebrates is influenced by chemical cues from microbial films, algae

(heterospecific) and conspecific adults; and by physical factors, such as

water flow, Iight and surface texture (Pawlik, 1992). Microbial films

induce larval metamorphosis of invertebrates such as bryozoans

(Brancato and Woollacott, 1982), hydrozoans (Leitz and Wagner, 1993),

polychaetes (Unabia and Hadfield, 1999), abalones (Kawamura and

Kikuchi, 1992), sea cucumbers (Ito and Kitamura, 1997) and sea urchins

(Ito, 1984), but has no effect on barnacles (Maki et al., 1988). Alnong

these organisms, bryozoan, hydrozoan and polychaete larvae

metamorphose on a clean surface, whereas for abalones and sea urchins,

the presence of microbial films is essential. In our study; we showed

the importance of microbial films in the larval metamorphosis of

Pseudocentrotus depressus and Anthocidaris crassispina.

In the sea, metamorphosis (%) of P depressus larvae increased

gradually with immersion period (Fig. 2.4), whereas the larval

metamorphosis of A. crassispina showed a bell-shaped response curve

(Fig. 2.5). Difference in larval response may be influenced by the film

components and growth rate of film, which reached the peak within a

shorter period during the hot season (May to August) than during the

cold season (October to May). We hypothesize that P depressus larvae

23

may also conform to the bell-shaped response curve if the immersion

period is extended beyond the 25-day period tested.

In the tank films, metamorphosis of P depressus larvae increased

linearly with diatom density ranging from 2.0xl04 to 6.0xl05 cells cm~

(Fig. 2.6), although the diatom-based films showed low

metamorphosis-inducing activity for larvae of A. crassispina (Fig. 2.7).

This pattem coincided with the data of Saga Prefecture (Ito et al. , 1991).

These results suggest that the periphytic diatoms on the tank film could

promote the larval metamorphosis of P depressus, but may be less

important in A. crassispina . Moreover, co-existing bacteria on the film

may also influence the larval response for both species of sea urchins.

There may be a difference between these two sea urchin species in

larval response towards microbial films, although the experiments were

conducted in two different seasons (Fig. 2.6, 2.7). A comparison of

results from larval assays simultaneous conducted in May (Fig. 2.8)

indicates that P depressus showed higher rate of metamorphosis than A.

crassispina in response to diatom-based films in the tank. This

difference seems to be related to their natural habitats P d . . epressus

juveniles are found in deep (8-12 m) rocky subtidal areas (Imai and Arai,

1994), whereas A. crassispina live in shallow (0.5-4.0 m) intertidal field

dominated by foliose coralline algae (Imai, 1980a, 1980b; Tsuji et al. ,

1989). Different habitats may influence larval response to certain

chemical cues in their environment.

/

24

　　　　The　findings　repOrted　hereprovide　initial　information　on　the　role　of

microbial　films　developed　in　the　sea　and　in　tank＄cult皿ing　the　periphytic

diatoms，on　induction　of　larval　metamorphosis　in　two　species　of　sea

urchins，R勿r63s〃5and乱c7σss剛nα．

25

CHAPTER - 111

The biological factors of diatom-based film for larval

metamorphosis of the sea urchins, Pseudocentrotus depressus and

Anthocidaris crassispina

III - I Introduction

In mass-production of juveniles of sea urchin, sea cucumber and

abalone, diatom-based film propagated on plastic plates ("nami-ita") in

5- to 15-tonne tanks is used to induce larval metamorphosis (Ito et al. ,

1991; Kawamura and Kikuchi, 1992; Kawahara, 1996; Ito and Kitamura,

1997). Diatom-based film is mainly composed of several species of

diatoms and multispecies of bacteria, but the role of either diatom or

bacteria in the induction of larval metamorphosis remains unclear.

Chapter - 11 mentioned that the diatom-based films in the

mass-production tanks induced the larval metamorphosis of

Pseudocentrotus depressus and Anthoc ~l ' l ans crassispina even though

larval response differed between the two species. That is, diatom-based

films formed in the tanks could promote larval metamorphosis in P

depressus but were less important in A. crassispina.

In the present study; in order to clarify the role of diatoms and

26

bacteria as biological factors of metamorphosis in larvae of sea urchins,

we investigated the effect of variously treated laboratory-cultured

diatom-based films on the metamorphosis of P depressus and A.

crassispina larvae. The effect of diatoms and bacteria on larval

metamorphosis was also examined using diatom-based films cultured in

the presence of germanium dioxide or antibiotic mixture. In addition,

we evaluated the metamo h rp osis-inducing activity of five periphytic

diatom species isolated from the films.

III - 2 Materials and methods

Diatom-based f'l 1 ms were cultured in 200-ml beakers (Fig. 3.1).

The seed film was obtained from the filmed plastic plates in the 5-tonne

tanks of the Nagasaki City Fishery Center, Nagasaki, Japan, and cultured

for several days under laboratory conditions (14:10 LD cycle;

4,000-7,000 Lux; 23'C) in 50-ml flasks containing 30 ml modified

Erdschreiber culture medium (l~:tamura and Hirayama, 1984). The

film was then scraped off the inner surface of the flask, thereby

suspending it in the medium. One milliliter of this suspension,

containing 2.0-5.0xl04 diatom cells, was inoculated into each

experimental beaker containing 100 ml of the medium. The beakers

were left under the conditions mentioned above for 5 days to allow

27

辮鰯～～～肇～レ

　　～

　　～”Nami－itバco飢ed　with

オ！periphyticdねt・ms　　5

　　～

Eldschreibe1＋vitamins（EV）

14：10LD　cyclel4シ000－7，000Luxl230C

篇

Day　O

100％EV

Day1雛圏聾斎麺醗→（顯

D＆y2　　　　　　　Day4

50％EV FSW

Multispecies

dia重Qm　and

　bac⑯ria

FSW

Fig・3・LDiat・m－basedfilm？ulturedinthelab・・縫t・r弘

28

development of film. The culture medium was changed to 50%-

diluted medium after 2 days and to filtered seawater (FSW, WhatmanR

glass fiber filter, GF/C; I .2 um) after 4 days. Periphytic diatoms, viz.

Navicula, Nitzschia, Amphora, Achnanthes and Cocconeis dominated

this cultured film. Each beaker was refilled with 100 ml FSW prior to

larval assays.

In a parallel experiment, variously treated diatom-based films were

prepared. Diatom-based film was scraped off one beaker, resuspended

in 100 ml FSW, and filtered through a glass fiber filter (WhatmanR,

GF/C). The filter with diatoms (hereafter referred to as "diatoms on

filter") was placed on the bottom of a beaker, which was then filled with

FSW and subjected to larval assay! On the other hand, the filtrate was

poured into a clean beaker to allow the development of bacterial film

(hereafter referred to as "bacterial film") and subjected to larval assay

(Fig. 3.2). A bacterial film with a density of 6.0xl05 cells cm~ was

verified to have formed on the bottom of the beakers within 2 h after the

commencement of assays.

Variously treated diatom-based films were also subjected to larval

assays. The first treatment was antibiotic treatment. Diatom-based

films were treated with FSW containing an antibiotic mixture (0.5 mg

ml~1 of penicillin G potassium and 0.01 mg ml~1 of streptomycin

sulphate) and subjected to larval assay. These antibiotics were

purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

29

Scraped film from a beaker

Film suspension in 100 ml FSW

Diatoms on filter

GF/C filter

"~~!~=~~~~~~-~_~~~ - ~!.~~!I~~~ ~~~

.~'_ ~~"=~~_=~~~-}~~~-S~~=.~~~'~. ~~~

~. ~=~-

Fig. 3.2.

Bacterial film

Preparation of diatoms on GF/C filter and

bacterial film developed from filtrate.

30

The specified concentrations of these antibiotics had been verified to

have no toxic effect on larvae (own unpublished data).

The effect of diatoms and bacteria on larval metamorphosis was

examined by using diatom-based films with germanium dioxide (Ge02)

or antibiotic mixture added in the film culture. For this experiment,

diatom-based films were cultured in 200-ml beakers containing 100 ml

of culture medium and cultured for 5 days under the same condition as

mentioned above (Fig. 3.1). Germanium dioxide (6.0 ug ml~1) to

depress the diatom density or antibiotic mixture (3.0 mg ml~1 of

penicillin G potassium and 0.06 mg ml~1 of streptomycin sulphate) were

added during the film culture period. The culture medium was changed

to 50%-diluted medium after 2 days and to FSW after 4 days with each

reagent, respectively! Each beaker was refilled with 100 ml FSW prior

to larval assays.

Five species of periphytic diatom (Achnanthes, Amphora,

Cocconeis, Navicula and Nitzschia) were successfully isolated from the

diatom-based film and were evaluated f or metamorphosis-inducing

activity in larvae. Each species of diatom was cultured from a single

cell isolated from a diluted film suspension under the stereomicroscope

using a fine tip capillary pipette. Each type of unialgal diatom film was

cultured in beakers using the same method described above for

diatom-based films (Fig. 3.1) and was subjected to larval assay. Larval

assays were conducted as described in Chapter - 11 (Fig. 2.2).

31

Throughout this study, enumeration of diatoms and bacterial

densities were counted from filmed glass slides (38x26 mm) prepared in

a similar manner as filmed beakers. Mean diatom and bacterial

densities were taken from twenty random spots on each glass slide and

were counted using a light microscope at 200x magnification and a

phase contrast microscope at 400x magnification, respectively.

All data are expressed as means (:!:SD). Differences between

control and treatments were analyzed using one-way ANOVA followed

by Tukey's multiple comparison test. Results were assessed significant

at P<0.001 .

III - 3 Results

Throughout all the experiments, the metamorphosis (%) of


crassispina in clean beakers containing only FSW (negative control) was

always O%. By contrast, the diatom-based films (positive control) that

consisted of multispecies periphytic diatoms and bacteria, showed larval

metamorphosis, ranging from 87% to 95% in P depressus and from 5 1 %

to 79% inA. crassispina.

The metamorphosis-inducing activities of the diatoms on filters,

the bacterial films and the diatom-based films treated with antibiotics

32

during the assay for both sea urchins are shown in Fig. 3.3 and 3 .4. In

P depressus (Fig. 3.3), the diatom-based films (positive co~trol) induced

95 % metamorphosis, while the diatoms on filters induced 67% Iarval

metamorphosis. However, no metamorphosis occurred on bacterial

films. Anthocidaris crassispina (Fig. 3.4) showed a response similar to

that of P depressus. Diatom-based films (positive control) induced

5 1 % metamorphosis, while the diatoms on filters induced 22% Iarval

metamorphosis. Bacterial films also did not induce larvae of A.

crassispina to metamorphose. In both species of, sea urchin, the

diatoms on filters showed lower activity than the diatom-based films

(p<d.OO1).

In assays with P depressus, the bacterial density of film treated

with antibiotics was 6.9xl06 cells cm~ while that of non-treated film

(positive control) was 7.1xl06 cells cm~2 24 h after the start of assay.

Thus, treatment with antibiotics did not reduce bacterial density; but it

did drastically reduced the inducing activity of diatom-based films (Fig.

3.3, 3.4). Assays employing larvae ofA. crassispina showed the same

trend. The rate of metamorphosis decreased to 3% and 18% in P

depressus and A. crassipina, respectively.

33

'~ ~~ee

¥J

O ~l ~l

O ~ Q) ~:

100

80

60

40

20

O

diatom-based f i Im

(positive control)

e

a

diatoms

on f ilter

bacterial antibiotics-f ilm treated

diatom-based f ilm

108 ~_

70S 10 ca

=t o 106 o ~ ~ 10s ~s~va

(D ~1

104 ~i::

o 103 *oe8

~ 11 102 ~

~ 10 _oee

,*

1 O


diatom-based films, diatoms on filters, bacterial films

and antibiotics-treated diatom-based films (0.5 mg ml~1

of penicillin G potassium and 0.0lmg ml~1 of strepto-

mycin sulphate) during the assay. Error bars indicate

:b SD of nine replicates except for control (n=18).

[1 : bacterial density; I : diatom density; horizontal

line: O% metamorphosis

34

~ ~~.'

~ ~:lOa:I

~ O ~O

:~

IOO

80

60

40

20

O

e

l a:

s

I

diatom-based diatoms f ilm on

(positive control) filter

bacterial antibiotics-

film treated diatom-based

f ilm

108 ~,~

'~

107 o gD

~ -o 106 o ,~ ~?

105 *F~uD

o ~:'

104 ~~:.::

o ~' In3 o lu cs

~ ~! 102 ~

~ 10 o *, Gs

,* 1 C~

Fig. 3.4. Larval metamorphosis ofA. crassispina in response to diatom-based films, diatoms on filters, bacterial films

and antibiotics-treated diatom-based films (0.5 mg ml~1

of penicillin G potassium and 0.0lmg ml~1 of strepto-

mycin sulphate) during the assay. Error bars indicate

:t SD of nine replicates except for control (n= 1 8).

[] : bacterial density, I : diatom density, horizontal


35

In diatom-based films cultured without addition of germanium

dioxide (Ge02), nor of antibiotic mixture (positive control), mean

metamorphosis rate of P depressus and A. crassispina were 87% and

79%, respectively (Fig. 3.5, 3.6). For films cultured with Ge02,

metamorphosis of P depressus was reduced significantly to 35%

(P<0.001) when diatom density was reduced by two orders of magnitude

(1.0xl03 cells cm~ , compared to the positive control film, 4.6xl05 cells

cm~2) (Fig. 3.5). Bacterial density on the Ge02 film was similar to that

on the positive control film. In contrast, metamorphosis-inducing

activity was reduced drastically to 9% (P<0.001) in film cultured with

antibiotic mixture, and bacterial density was reduced by one order of

magnitude (5.3xl05 cells cm~ , compared to the positive control film,

5.6xl06 cells cm~2). Diatom density on antibiotic films was similar to

that on the positive control film.

A. crassispina (Fig. 3.6) also showed similar response to that of P

depressus. The metamorphosis of film cultured with Ge02 Was reduced

significantly to 52% (P<0.001) when diatom density was reduced by two

orders of magnitude (2.4xl03 cells cm~ , compared to the positive control

film, 3.2xl05 cells cm~2). Larval metamorphosis was reduced to 16%

(P<0.001) in film cultured with antibiotic mixture, when the bacterial

density was reduced by an order of magnitude (4.8xl05 cells cm~ ,

compared to the positive control film, 4.2xl06 cells cm~2).

36

1 OO

80

~ ~~.'

~ '"* 60 ~i

~ ~o 40

~:

20

O

diatom-based f ilm

Gpositive control)

A

e

I ~

B

c

+ G, e02 + antibiotic

mixture

108 ~-

70~ 10 GQ

~ ~ 106 ~

~ .* 10s ~ o 1:'

104 ~!:'

o 103 ~o,s

~ ~' 102 ~

~ 10 o ~ es

.* 1 C:i


diatom-based film cultured with germanium dioxide,

Ge02 (6.0 u g ml~1) and cultured with antibiotic mixture

(3 .O mg ml~1 of penicillin G potassium and 0.06 mg ml~1

of streptomycin sulphate). Error bars indicate i SD of

eighteen replicates. Values not sharing common letters

are significantly different (P<0.001). [] : bacterial

density; I : diatom density

37

100

80

~ .¥'

~ '~ 60 ~!

~ ~o 40

:~

20

O

diatom-based f ilm

(positive control)

A a:

~ B

c

+ Ge02 + antibiotic mixture

108 ~_ '~

107 ~

~ ~ o 106 ~'o

~ 10s ~ o ~:'

104 ~i::

_o 103 cso

~ ~' 102 ~

~ 10 -eeo

,* 1 C:I


diatom-based film cultured with germanium dioxide

Ge02 (6.0 u g ml~1) and cultured with antibiotic mixture

(3.0 mg ml~1 of penicillin G potassium and 0.06 mg ml~1

of streptomycin sulphate). Error bars indicate :1: SD of

eighteen replicates. Values not sharing common letters

are significantly different (P<0.001). [1 : bacterial

density; I : diatom density

38

　　　　None　of　the　five　species　of　diatom　isolated　from　the　diatom－based

film（、4chnαn孟hε＆。4醒ρho鵜Cocconε鶴ハ砂v∫cμ伽and荒砿sch∫α）induced

larval　metamorphosis　in　either　gfthe　two　species　ofsea　urchin（Fig．3．7，

3．8）；nevertheless，the　metamorphosis　rate　of　R喫ρrε3sμ3and1生

c■αssゆ功α　exposed　to　multisp停cies　diato血一based　films　（positive

“ontrol）were89％and62％，respective1》乙

39

F, ~~ee

~'

O ~ el O ~ Q) ~:

1 OO

80

60

40

20

o

e

I

e

l

e

l

e

l

e

l

~ l~ ~ q:1,,:!o

1$~l Q)O ~o ~o l> ~~~ ~!oca

~~

e~ C':I

~*~

~:

~ ~~!)

,~:

v:~

L~

~ E~~

~:

pl Ga EQ .~ ~) ~: q) ~) ~) Cb ~J

~l co

_~~$

~: ~b .~ ~~~

Ga~

.~ ~~)

:~!._.~e

~

108

~~ '~

107 o ~,D

~ ~ In6 o u~ ~ 105 '~

o =1 104 ~j

.F: 103 *o'L)c:l

~ 102 ~' ~ ~

10 _o~s

.~ 1 O

Fig. 3.7. Larval metalnorphosis of P depressus in response to

five species of periphytic diatoms isolated from

diatom-based film. Error bars indicate :1: SD of .

eighteen replicates. [] : bacterial density, I : diatom

density; horizontal line: O% metamorphosis

,

40

~ ~~.'

~ ~

~::

~ o ~O

~:

100

80

60

40

20

o

e s

l

5

l

e

l

e

l

e l

~(:r ~::O -S! ~'$::

OO ~O ~O l .> ~:i-~,**

Oc'D ~'eS O '~ie'

eo~

~:~

~;

~: ~~:cL)

~~

~$::*

L~$

~ ~~~

~:

olE'e

ee .~ qD ~: cb ~) c!)

s ~)

O1 C':I

'~~~$

~: ~) .~ ~~~

pl ca-

.~ ~~ ~)

.1~.__N~':

~

108 ~_

70~ 10 c'b

~ -68 10 -~ .* 10s ~ o 11

104 ~!_oE1

103 ~cso

JD It

102 ~

~ 10 -o~5

.~ 1 C:I


five species of periphytic diatoms isolated from

diatom-based film. Error bars indicate :!: SD of

eighteen replicates. [1 : bacterial density~ I : diatom

density, horizontal line: O% metamorphosis

41

-~

III - 4 Discussion

Diatom-based films have been used to induce larval metamorphosis

during mass-production of sea urchins juveniles (Ito et al. , 1991;

Kawahara, 1996). In the present paper, the results of a preliminary

study to elucidate the roles of diatoms and bacteria on larval

metamorphosis of Pseudocentrotus depressus and Anthocidaris

crassispina have been reported.

Attempts were made to evaluate separately the

metamorphosis-inducing activity of diatoms and bacteria from

laboratory-cultured diatom-based film. Diatoms on the filter induced

metamorphosis in larvae of both species, even though the activity was

significantly lower than that of the control (Fig. 3 .3, 3.4). By contrast,

bacterial films did not induce metamorphosis in larvae of either species.

These results suggest that bacteria alone cannot induce larval

metamorphosis and that diatoms play a major role in the induction of

larval metamorphosis in these sea urchins. However, it should be noted

that the metamorphosis-inducing activity of the diatoms on filter showed

a significant decrease compared to the film; therefore, it can be assumed

that a metamorphosis-enhancing factor in diatom-based film passed

through the filter. It is also possible to hypothesize that the mixture of

diatoms and bacteria provide a synergistic effect for the

metamorphosis-inducing activity of diatom-based film. A different

42

explanation could be a difference in the configuration of the diatom films

cultured on beakers and those collected on filters. This reduction in

metamorphosis-inducing potency requires further investigation.

In the present investigation, treatment with antibiotics (penicillin

and streptomycin) drastically reduced the metamorphosis-inducing

activity of diatom-based films, even though bacterial growth was not

controlled. The concentrations of antibiotics used in this experiment

should had no adverse effect on the sea urchin larvae (own unpublished

data); I suggest that the antibiotic mixture may have affected the

condition of the diatom-based films.

The larvae of P depressus 'and A. crassispina showed similar

response towards the film cultured with germanium dioxide and

antibiotic mixture (Fig. 3.5, 3.6). For both species, reduction in

bacterial density had a greater effect on the metamorphosis-inducing

activity of films than did reduction in diatom density; with a drastic

decrease of 80% to 90% in the former, compared to 35% to 60% in the

latter. Based on this results, it is possible that the

metamorphosis-inducing activity of diatom-based film for larval

metamorphosis of these sea urchins may be due to the synergistic effect

of diatoms and bacteria as mentioned earlier (Fig. 3.3, 3.4).

None of the five isolated species of periphytic diatom induced

metamorphosis in larvae of P depressus and A. crassispina (Fig. 3.7,

3 .8). This suggests that no single diatom species is directly involved as

43

an inducers of metamorphosis in sea urchin larvae. The elucidation of

the role of diatoms in combination with other factors requires further

investigation.

Although diatom-based films cultured in the laboratory induced

larval metamorphosis in both P depressus and A. crassispina, the

response differed between the two species; higher metamorphosis (%)

was observed in P depressus than in A. crassispina . This observation

is consistent with the results shown in Chapter - II; that is, diatom-based

films formed in the sea urchin juvenile mass-production tanks promote

larval metamorphosis in P depressus but are less important in A.

crassl spma .

44

CHAPTER - IV

Characteristic of the chemical cue in diatom-based films for larval

metamorphosis of the sea urchins, Pseudocentrotus depressus and

Anthocldans crassispina

IV - I Introduction

As mentioned in the previous chapters, competent larvae of sea

urchins Pseudocentrotus depressus and Anthocidaris crassispina must be

able to detect the metamorphosis-inducing cue released by suitable

inducers such as microbial films in the sea, diatom-based films formed in

the sea urchin juveniles mass-production tanks and laboratory-grown

f ilms .

A number of researchers have studied the chemical cues for larval

metamorphosis of sea urchins derived from algae, although real chemical

cues have been rarely identified. Free fatty acids, such as

eicosapentaenoic acid and arachidonic acid extracted from coralline red

alga Corallina pilullfera, were reported to induce larval metamorphosis

of P depressus and A. crassispina (Kitamura et al., 1992; 1993).

Glycoglycerolipids from green alga Ulvella lens induced larval

metamorphosis of Stron8ylocentrotus intermedius (Takahashi et al.,

45

2002). A water-soluble complex of the sugar floridoside and isethionic

acid isolated from red alga Delisea pulchra triggered metamorphosis in

Holopneustes purpurascens larvae (Williamson et al. , 2000).

Dibromomethane that was detected in the seawater, where U. Iens or

coralline red algae were previously cultured, induced larval

metamorphosis of S. nudus (Taniguchi et al. , 1994). In larvae of P

depressus and A. crassispina, high concentration of synthetic

dibromomethane induced metamorphosis at 3,800 ppm and 2,200 ,ppm,

respectively (Koh et al., 1996).

An amino acid, L-glutamine also induced larval metamorphosis in

several sea urchins, Hemicentrotus pulcherrimus (Yazaki, 1995), P

depressus (Yazaki and Harashima, 1994) and S. intermedius (Naidenko,

1996). Naidenko (1996) also reported that the epiphytic calcareous

alga Melobesia sp., colonizing the older sea grass Zostera marina,

induced metamorphosis of S. intermedius larvae and this alga contained

L-glutamine. Larvae of S. droebachiensis metamorphosed in response

to GABA-mimetic molecules contained in coralline red alga

Lithothamnion glaciale (Pearce and Scheibling, 1990).

In the present study, we attempted to elucidate the characteristics of

the chemical cue in the laboratory-cultured diatom-based films that

induced larval metamorphosis of P depressus and A. crassispina larvae.

The metamorphic activity of potential cues in film was evaluated after

heat, ethanol, hydrochloric acid, glutaraldehyde or lectin treatments.

46

IV - 2 Materials and methods

The nature of the chemical cues in laboratory-cultured

diatom-based films for larval metamorphosis was investigated by heating,

ethanol (EtOH), hydrochloric acid (HCl), glutaraldehyde and lectin

treatments, and subjected to larval assays (Fig. 4.1). Heat and lectins

treatments were carried out to determine whether the cue is heat-stable

and is a sugar-related compound. HCI and glutaraldehyde treatments

were done to check the induction activity of non-living films.

Diatom-based films were heated (30'C, 35'C, 40'C or 45'C) in a

water incubator for 30 min. Diatom-based films were also treated for

30 min with EtOH (1%, 5% or 10%) or HCI (0.1 N or 1.0 N). In the

glutaraldehyde (5%) treatment, diatom-based films were treated for 24 h,

then rinsed with FSW 3 times per day for 3 consecutive days. The

treatments of diatom-based films were also performed with the lectins

LCA (Lentil Agglutinin), SBA (Soybean Agglutinin), or WGA (Wheat

Germ Agglutinin) at 100 ug ml~1 concentration for 2 h. The lectins

were purchased from Wako Pure Chemical Industries, Ltd. (Osaka,

Japan). Prior to larval assays, all filmed beakers were rinsed with FSW

for 3 times and refilled with 100 ml FSW. Larval assays were

conducted as described in Chapter - 11 (Fig. 2.2).

Throughout this study; Iaboratory-cultured diatom-based film was

prepared as mentioned earlier in Chapter - 111 (Fig. 3.1). Diatom and

47

Treatment

Hea‡（30min）

　　300C，350C，400C，450C

EtOH（30min）

　　1％，5％，10％

HCl（30min）

　　0．1N，1．ON

Glutaraldehyde，5％（24h）

．恥ctins・100國m1（2h）

　　　LCA，SBA，WGA

rinsewith

FSW

Larval

assay

Fig．4．1。Summary　ofheat　and　chemical　treatments．

bacterial densities were enumerated as described in Chapter - 111.

Statistical analyses were done as mentioned in Chapter - 111.

IV - 3 Results

Throughout all the experiments, the rate of metamorphosis in


crassispina in negative controls was always O%. By contrast, the

laboratory-cultured diatom-based films (positive control) that consisted

of multispecies periphytic diatoms and bacteria, induced larval

metamorphosis greater than 90% in P depressus and from 61% to 85%

in A. crassispina . The diatom and bacterial density was determined

after heat, ethanol (EtOH), hydrochloric acid (HCl), glutaraldehyde or

lectin treatments mentioned below, and no significant difference was

observed compared to the untreated film.

Larval metamorphosis in both species of sea urchins exposed to

diatom-based films treated with heat is shown in Fig. 4.2 and 4.3. For P

depressus (Fig. 4.2), diatom-based films (positive control) showed 93 %

metamorphosis and the metamorphosis-inducing activity decreased

drastically to O% at 40'C and 45'C (P<0.001). ForA. crassispina (Fig.

4.3), the larval response was similar to P depressus. Diatom-based

films (positive control) showed 61% metamorphosis and the

49

'~ ~:~ee

'~'

~:O

~ O ~ o :~

1 OO

80

60

40

20

O

diatom-based film

(positive control)

~

E: i l~lli '!'

.

~=

al l

,i

~~i

ll

i ll l lr '

'l

~' ji~ii,

.I ~ta

~l

'l:

I ~ 'ii

~l

':~: ~"' il 'i~ii

iiil

ii ~

l --

I~ i:1 :

300C 35"c 400C 450c

108 ~,~

'~

107 o c'a

-~ o 106 o ,J ~ ." 105 ~ o =1

104 .ce~~

~a)

In3 o lu ~~ ~'

102 ~

~ o 10 ~~ Gs .* C:I

1

Fig. 4.2. Larval metamorphosis of P depressus in response to diatom-based film treated with heat (300C, 350C, 400C

and 450C). Error bars indicate :!: SD of eighteen

replicates. [] : bacterial density; I : diatom density;

horizontal line: O% metamorphosis

50

l~* ~~OO

~J . Ga_,

O ~ 81 O ~Q)

~:

100

80

60

40

20

o

~l

jii~i

~ l!i

~pl

jl

j~~i*

lli~i*,~

~lpi~

' ~~

j~i~~

j~i~i~

~~~,':1!

~i

l~

~jr;'~i

.~i

p*

~ .~

s

l

di~i#

jr'

:E

l

diatom-based 300C film

(positive control)

35'c 40'c 450C

108 ~,_.

'~

107 o c':I

~ ~ o 106 ~~

~ 5 '~uD 10 ,sl *Q) ~)

104 .~ ,~ ~o

103 ~c~o

=1 102 ~

~ 10 o ~' ee

.* O 1


diatom-based film treated with heat (300C, 350C, 400C

and 450C). Error bars indicate :1: SD of eighteen

replicates. [] : bacterial density; I : diatom density;

horizontal line: O% metamorphosis

51

metamorphosis-inducing activity decreased drastically to 10% and O% at

40'C and 45'C, respectively (P<0.001).

The metamorphosis-inducing activity of diatom-based films treated

with EtOH, HCI or glutaraldehyde is shown in Fig. 4.4 and 4.5. For P

depressus (Fig. 4.4), metamorphosis was also reduced significantly to

50% in 5 %-EtOH and to 7% in 10%-EtOH-treated diatom-based films

(P<0.001). By contrast, Iarvae did not metamorphose in response to

HCI (0.1 N or I .O N) or 5% glutaraldehyde treated diatom-based films

(P<0.001). For A. crassispina (Fig. 4.5), the larval response was

similar to P depressus except in diatom-based films subjected to

5 %-EtOH treatment. In A. crassispina, metamorphosis decreased

significantly to 22% only in 10%-EtOH-treated diatom-based films

(P<0.001), but not in 5%-EtOH. Diatom-based films treated with HCl

or glutaraldehyde did not induce metamorphosis (P<0.001).

On the other hand, Iectin (LCA, SBA or WGA) treatments did not

affect the metamorphosis-inducing activity of diatom-based films either

in P depressus (Fig. 4.6) or A. crassispina (Fig. 4.7).

52

'~ ~~eo

'v'

uDO

~:1

B~

O ~ 'L)

~:

1 OO

80

60

40

20

o

~ dll~:'

d ~ ~i:

p lpll~I~

~ B S g!: 9: l

~~~!f*

~ d~::

i!r

~l~!' E

!!1li~' !!!~

illli*' '

illii~:~" l~~~

~l~llii ~i!l ~

'd:**!!*

l~"' .*.

~~ii~' jpii~i

.'~~d '~k ~･*

diatom- 1% 5% 10% 0.1 N based

EtOH

108 -~~

70 10 va --o 60 10 -~~

10s c'aoF~

~' 104 ~I,~

o ~ 103 ~c:'o

~1 102 ~

~ o 10 -~3 .* C:I

1

1.0 N 5% glutar-

aldehyde

(positive control)


diatom-based film treated with EtOH (1%, 5% and

10%). HCI (0.1 N and 1.0 N) and glutaraldehyde (5%).

Error bars indicate :1: SD of eighteen replicates.

[] :, bacterial density; I : diatom density; horizontal


53

~ ~~'e

,J

~ ~:I

~ O ~ o :~

100

80

60

40

20

O

o o

~ rr

llllk!i'

'~l jii~

* e~-*

~! ..~

piF'

5

ptli

lliii~

~l~i~

'I:1 ~

i~:'

~

~L

l

~ ~.

j~i

~!-~

,~ ~

5:

l

~

l

~:

l

diatom-

based film

(positive control)

1%

108 -'~

107 ~

-~ o 106 ~

~ .* 105 ~ o ~:l

104 ~i::

o * 103 ~cso

~1 102 ~

~ 10 o ~ ee

.-~l 1

5% 10% 0.1 N 1.0 N 5% glutar-

EtOH HCI aldehyde

Fig. 4.5. Larval metamorphosis ofA, crassispina in response to

diatom-based film treated with EtOH (1%, 5% and

10%), HCI (0.1 N and 1.0 N) and glutaraldehyde (5%).

Error bars indicate -+ SD of eighteen replicates.

[] : bacterial density; I : diatom density; horizontal


54

l~ ~~ee

,~

O ~:l

~1

O ~ O ~:

100

80

60

40

20

O

~ ~~:ll! Il!

pt~iPl ~i~i~

~l 'i

~" ~i

pi~

l#~~*

ji~

pt!~ pii~~~ ~

~i!c

~iii:~gi ~li~~,j 'i

lll~i~,

~1 j~~ ~~

jpi '

diatom-based f ilm

LCA SBA WGA

108 ~,_,

'~

107 ~

~ ~ o 60 10 -~ 105 ~ o '1'

104 ~i.*

h o 1. 103 oc:'

~) ~1

102 ~ ~

10 o ~ 's

.-C:I 1

(positive control) '


diatom-based films treated with lectins (LCA, SBA

and WGA). Error bars indicate :b SD of nine replicates.

[] : bacterial density, I : diatom density

55

'~. ~~ee

~' aaO

~:i

el

O ~ Q) ~:

1 OO

80

60

40

20

O

diatom-based f ilm

(positive control)__

~ l!Ii

llil~:

i

ppi~ li l~~

jllil

j ~~! ~ ~1lpi=

l

'. jil

i~:

~i~: l~~ jipii l.r

t

i

piii~ pti

d' '

jlll~1i

~

LCA S BA WGA

1 08

~'~ '~

107 ~

~ 106 ,JoQ)

~ ~ 10s '~

o 11 104 ~!,~

_o 103 cso

~ lc;

102 ~

S 10 o ~ Ge

.-1 C:I


diatom-based films treated with lectins (LCA, SBA

and WGA). Error bars indicate :t SD of nine replicates.

[] : bacterial density, I : diatom density

56

IV - 4 Discussion

Diatom-based films have been used to induce larval metamorphosis

during the mass-production of sea urchins juveniles (Ito et al. , 1991;

Kawahara, 1996). In this section, preliminary study was done to

investigate the nature of the chemical cue in diatom-based films on larval

metamorphosis of Pseudocentrotus depressus and Anthocidaris

crassl spma .

The larval response of P depressus and A. crassispina towards

laboratory-grown diatom-based films treated with heat (Fig. 4.2, 4.3),

ethanol (EtOH), hydrochloric acid (HCl) and glutaraldehyde (Fig. 4.4,

4.5) clearly shows that the cue is susceptible to degradation.

Glutaraldehyde, HCI and heat (above 40'C) treatments all completely

destroyed the. metamorphosis-inducing activity of diatom-based films.

This suggests that the live films possess an unstable cue that was easily

inactivated by heat. By contrast, Iectin treatment did not affect the

metamorphosis-inducing activity of diatom-based films (Fig. 4.6, 4.7),

which suggests that sugar-related compound do not play a main role in

the metamorphosis.

Metamorphosis-inducing compounds previously reported for sea

urchins are sugar floridoside and isethionic acid (Williamson et al. ,

2000), eicosapentaenoic acid (Kitamura et al. , 1993) and

glycoglycerolipids (Takahashi et al., 2002), which are relatively stable at

57

400C．　Thus，the　cues　in　diatom－based　film　may　be　difOerent　from

pleviously　reported　ones．Dibromomethane（Taniguchiαα乙，1994）is

a　possible　candidate　as　an　hlducing　cue，but　a　high　concentration（2，200

to3，800ppm）is　needed　to　induce　larval　metamorphosis　of　these　two

species　of　sea　urchin（Kohα‘zJ．，1996）．

58

CIIAPTER - V

GENERAL DISCUSSION AND CONCLUSIONS

In Japan, diatom-based film had been used in aquaculture to

mass-produce juveniles of sea urchin (Kawahara, 1996), sea cucumber

(Ito and Kitamura, 1997) and abalone (Kawamura and Kikuchi, 1992;

Kawamura, 1996) to sustain the wild stock. Mass-production of

economically important species of sea urchin juveniles (Anthocidaris

crassispina , Hemicentrotus pulcherrimus, Pseudocentrotus depressus, S.

intermedius, S. nudus) is being done annually (Kitamura et al. , 1993) by

inducing metamorphosis of competent larvae using plastic plates

("nami-ita") covered with periphytic diatoms. These studies indicated

the importance of diatom-based films in larval metamorphosis of sea

urchin, though the role of periphytic diatom and bacteria co-existing in

the film remains unknown.

In the present study; the metamorphosis-inducing activity of

microbial films formed in the sea and diatom-based films formed in

periphytic diatoms culture tanks on the larval metamorphosis of two

species of sea urchins P depressus and A. crassispina were investigated

(Chapter - II). Attempts were also made to separately evaluate the

metamorphosis-inducing activity of diatoms and bacteria from the

laboratory-cultured diatom-based films (Chapter - 111). Moreover, a

59

preliminary study was done to understand the nature of the chemical cue

in diatom-based films on larval metamorphosis of these two sea urchins

(Chapter - IV).

As has been detailed in Chapter - II, microbial films in the sea

showed that metamorphosis of P depressus larvae increased gradually

with immersion period, whereas the larval metamorphosis of A.

crassispina showed a bell-shaped response curve. Difference in larval

response may be influenced by the film components and the growth rate

of film, which reached the peak within a shorter period during spring and

summer (A. crassispina) than during the autumn and winter season (P

depressus). I hypothesize that P depressus larvae may also conform to

the bell-shaped response curve if the immersion period is extended

beyond the 25-day period tested.

In the tank films, metamorphosis of P depressus larvae increased

linearly with diatom density ranging from 2.0xl04 to 6.0xl05 cells cm~ ,

although the diatom-based films showed low metamorphosis-inducing

activity for larvae of A. crassispina. This pattern for P depressus

coincided with the data of Ito et al. (1991). These results also suggest

that the periphytic diatoms in the tank film could promote the larval

metamorphosis of P depressus, but may be less important in A.

crassispina (Chapter - II).

A comparison of results of larval assays done simultaneously in

May indicates that P depressus showed higher metamorphosis than A.

60

crassispina in response to diatom-based films in the tank. This

difference may be related to their natural habitats. P depressus

juveniles are found in deep (8-12 m) rocky subtidal areas (Imai and Arai,

1994), whereas A. crassispina live in shallow (0.5-4.0 m) intertidal field

dominated with foliose coralline algae (Imai, 1980a; 1980b; Tsuji et al. ,

1989). In other words, the microbial films on rock surfaces act as

metamorphosis inducer for larvae of P depressus, while the cue from

algae induces larval metamorphosis ofA. crassispina (Chapter - II).

In the Chapter - 111, diatom-based films grown under laboratory

conditions also induced larval metamorphosis of both sea urchins.

Furthermore, diatoms on filters induced metamorphosis in larvae of both

species, even though activity significantly decreased as compared to the

control group. By contrast, the bacterial films (filtrate) did not induce

metamorphosis in larvae of both species, suggesting that bacteria alone

cannot induce larval metamorphosis and that diatoms play a major role

in the induction of larval metamorphosis in sea urchins. However, it

should be noted that metamorphosis-inducing activity of the diatoms on

filters significantly decreased. It can be assumed that either a

metamorphosis-enhancing factor in diatom-based film passed through

the filter or a difference in surface configuration of diatom films between

those grown on beakers and those collected on filter may have affected

results.

It was also found that the antibiotic mixture (penicillin and

61

streptomycin) during larval assay destroyed or erased the

metamorphosis-inducing activity in diatom-based films, even though

bacterial density was not significantly decreased. Hence, the result

suggests that antibiotic mixture could have affected the quality of

diatom-based film community (Chapter - 111).

The larvae of both sea urchins showed similar response towards the

diatom-based film cultured with germanium dioxide and antibiotic

mixture. Reduction in bacterial density had a greater effect on the

metamorphosis-inducing activity of films than did reduction in diatotn

density, with a drastic decrease of 80% to 90% in the former as

compared to 35% to 60% in the latter. All five isolated species of

periphytic diatoms did not induce metamorphosis of both sea urchins

larvae. This suggests that no single diatom species is directly involved

as an inducer of metamorphosis in sea urchin larvae (Chapter - 111).

As has been noted above, diatom-based films cultured in the

laboratory induced larval metamorphosis in both P depressus and A.

crassispina. However, response of larvae to the diatom-based films

differed between the two species in that higher metamorphosis was

observed in P depressus than A. crassispina . This observation suggests

that diatom-based films formed in the sea urchin juvenile

mass-production tanks could promote larval metamorphosis in P

depressus but were less important in A. crassispina . Based on my study,

it is possible to hypothesize that the mixture of diatoms and bacteria

62

provide a synergistic effect for metamorphosis-inducing activity of

diatom-based film (Chapter - 111).

Laboratory-cultured diatom-based films induced larval

metamorphosis and a preliminary study was done to investigate the

nature of the metamorphosis-inducing chemical cue in diatom-based

films for P depressus and A. crassispina (Chapter - IV). The cue was

found to degrade when treated with heat (40'C), 5%- and 10%-ethanol,

0.1 N hydrochloric acid and 5 % glutaraldehyde. These results suggest

that the films possess an unstable chemical cue that was very degradable

in heat and chemically unstable. By contrast, Iectin treatment did not

affect the metamorphosis-inducing activity of diatom-based films,

suggesting that the cue is not a sugar-related compound. Previously

reported metamorphosis-inducing compounds for sea urchins were a

complex of the sugar floridoside and isethionic acid (Williamson et al. ,

2000), eicosapentaenoic acid (Kitamura et al. , 1993), and

glycoglycerolipids (Takahashi et al., 2002); all of them are relatively

stable at 40'C. Treatment of diatom-based films by ethanol (5% and

10%) may have affected the activity of diatoms and bacteria, and thus

resulting in the decrease in its metamorphosis-inducing activity.

Dibromomethane (Taniguchi et al., 1994) may be a possible candidate as

inducing cue, but high concentration was needed to induce larval

metamorphosis of these two species of sea urchins (Koh et al., 1996).

Thus, the cues in diatom-based film may be different from previously

63

reported ones.

Based on the overall findings in the present study, the microbial

films formed in the sea triggered laryal metamorphosis of P depressus

and A. crassispina although the pattern of larval response differed. In

diatom-based film formed in tanks, metamorphosis of P depressus

larvae increased linearly with diatom density; but showed low inducing

activity for larval metamorphosis of A. crassispina . The difference in

larval response of these species may be related to their natural habitat.

It is also possible to hypothesize that the mixture of diatoms and bacteria

provides a synergistic effect for the metamorphosis-inducing activity of

diatom-based film since bacteria alone or isolated periphytic diatoms

cannot induce larval metamorphosis. It is suggested that live films

possess an unstable cue(s) that was easily inactivated by heat and

sugar-related compounds do not play a main role in the metamorphosis.

The roles of periphytic diatoms and co-existing bacteria in

diatom-based film in the induction of larval metamorphosis require

further investigation. Moreover, the identification of the chemical

structure of the cues and the receptor sites in the larva are important for

understanding the mechanism and chemical ecology of metamorphosis

in sea urchin larvae. The findings reported here provide initial

information on the role of microbial films formed in the sea,

diatom-based films in periphytic diatom culturing tanks and in the

laboratory, on induction of larval metamorphosis in two species of sea

64

皿chins，R塵ρr8ssμs　and／i1．c7α55麹ρ∫nα．　These　in£ormation　may

improve　the　techniques　of　juveniles　mass－production　of　fisheries

important　species　of　ma血e　invertebrates　especially　sea皿chins　and

other　echinoderms．

65

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